US 20090305397 A1
A device for transporting at least one cellular entity during culture or maturation, the device comprising a substrate having one or more wells, said one or more wells being adapted to hold a cellular entity in a fluid, lid means for preventing entry or exit of the cellular entity from the one or more wells and fluid transport means connecting the one or more wells to enable flow of fluid or diffusion of chemical species. The apparatus alternatively or in addition comprise a module for transporting a payload at a controlled temperature, the module comprising an outer housing, an outer thermally insulating region, an inner thermally insulating region, and a heat sinking region located between the inner and outer thermally insulating regions, the inner thermally insulating region defining a cavity for receiving a payload, and heating means.
1. A device for culturing or maturing cellular entities, the device comprising a substrate having one or more wells, said one or more wells being adapted to hold a cellular entity, and lid means releasably-secureable to the substrate to prevent entry or exit of the cellular entity, wherein the device further comprises a source of a fluid and fluid transport means to feed the fluid from the source to the one or more wells in use.
2. A device for transporting at least one cellular entity during culture or maturation, the device comprising a substrate having one or more wells, said one or more wells being adapted to hold a cellular entity in a fluid, lid means for preventing entry or exit of the cellular entity from the one or more wells and fluid transport means connecting the one or more wells to enable flow of fluid or diffusion of chemical species.
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4. A device as claimed in any preceding claim in which the fluid consists of a gas and the fluid transport means comprises a gas permeable element.
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7. A device as claimed in claim any preceding claim in which the wells are adapted to prevent physical contact between cellular entities in adjacent wells while allowing chemical transport between the wells.
8. A device as claimed in any preceding claim in which the one or more wells are tapered to locate the cellular entity at a given location in the or each well.
9. A device as claimed in any preceding claim in which fluidic pathways are provided between a plurality of wells.
10. A device according to any preceding claim wherein the fluid transport means comprises a material which controls diffusion and/or convection of the fluid between wells.
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12. A device as claimed in any preceding claim including means for altering the composition of a liquid medium in the one or more wells with time.
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22. A device as claimed in any preceding claim further comprising a temperature sensor and temperature control means.
23. A device as claimed in any preceding claim wherein the device further comprises one or more fluidic channel(s) open to the or each well.
24. A device as claimed in any preceding claim further comprising a memory system.
25. Apparatus including a device as claimed in any preceding claim and a transport module adapted and arranged to control the operation of said device in transit.
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41. An apparatus for transporting a payload at a controlled temperature, the apparatus comprising an outer housing, an outer thermally insulating region, an inner thermally insulating region, and a heat sinking region located between the inner and outer thermally insulating regions, the inner thermally insulating region defining a cavity for receiving a payload.
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This invention relates to a system and method for culturing cells, oocytes, embryos, maturing ova or other cellular structures in vitro. It also relates to means for transportation of cells, ova, embryos, oocytes or other cellular structures or entities.
Various apparatus and methods are known for maturing ova and culturing embryos in vitro. In standard practice these processes are achieved using conventional tools such as pipettes for manipulation of an ovum or embryo, and Petri dishes to contain the ovum or embryo and maturation or culture medium. The ova or embryos are usually cultured in an incubator in conditions of controlled temperature and gas environment. They may be cultured singly or in groups, and for ova in particular, may be cultured in the presence of other cells, such as cumulus cells. Maturation or culture is often done in microdrops of medium in a Petri dish, the medium covered by an inert oil, the dish having gas access to the environment in the incubator. In some conventional maturation or culture procedures the volume of the medium environment in which the ovum or embryo is contained is important there is evidence in some methods that maturation and culture is more successful if several ova or embryos are present together in a small volume of medium. This autocrine effect is thought to result from trace chemical substances produced by a first ovum or embryo affecting the development of a second. However, it is also advantageous in certain circumstances to track the identity of individual ova or embryos and conventional apparatus in general does not allow the embryos or ova to be kept separate while allowing exchange of chemical substances between them. The well-of-wells (WOW) method of Vajta et al. as disclosed in WO 0 102 539 allows this to be done, but does not close the wells against exit of the embryos and so is not suitable for use in a transportable device.
The medium is usually buffered against changes in pH; this buffer may be based on bicarbonate/CO2, in which case the partial pressure of CO2 in the external gaseous environment is important, and it may be based in whole or part on other buffer systems, for example HEPES, in which case the gaseous environment may be less closely controlled or in some circumstances not controlled at all. The medium may be of nominally constant composition during maturation or culture, or may be changed, for renewed media of the same nominal composition, or a new medium to modify the medium conditions in order for example to assist or control the process of maturation or culture. In particular, in certain methods for culture of embryos it is known to be advantageous to culture the embryos initially in serum-free medium, changing to medium containing serum (often fetal calf serum, FCS) later in culture. In the case of maturation of ova, it is known that the progress of maturation may be controlled by addition of species to the maturation medium or their removal from it by replacing the medium with fresh medium. This may be particularly advantageous if the ova or embryos are to be transported during the maturation or culturing process, for example from a location at which the ova are harvested or the embryo created, and a second location where the ova might be used or the embryo implanted. Conventionally medium is changed by moving the ovum or embryo by pipetting from one medium to another, for example from one microdrop to another in a common culture dish. This uses simple apparatus but suffers from several disadvantages: the ova and embryos are delicate and can be damaged by pipetting; an amount of medium is necessarily transferred from one medium environment to another, which is significant especially in the small volume of a microdrop, and gives the possibility that substances from the old medium active at very low concentrations may be transferred into the new medium, unless sequential washing steps are used; the transfer process is slow and requires skilled personnel; and the transfer cannot be done remotely, so cannot be done in transit or outside a fully equipped laboratory setting.
In the description that follows reference will be made to culture of embryos as an example of the function of apparatus and description of the method. Many of the processes can also be applied to maturation of ova and culturing of cells or other cellular entities and it will be apparent to those skilled in the art how this application can be made, with appropriately chosen dimensions for the different size scales of embryos, ova and cells. Therefore the terms maturation and culturing, and ova and embryos and cells, are used interchangeably in the following and where convenient referred to collectively as >objects=. Where specific features of the invention apply to maturation of ova, or to culturing of embryos, this will be noted.
A number of apparatus and methods have been proposed to alleviate these and other problems in the conventional art.
Beebe et al. U.S. Pat. No. 6,193,647, U.S. Pat. No. 6,695,765 have proposed a system of approximately embryo-sized microchannels in which the embryos reside, being located at a constriction within the microchannels by entrainment in flow along the channels, that flow causing them to roll along the channel in contact with one of the channel walls. This apparatus achieves close control of the medium environment of the embryo, but suffers from the disadvantages, among others, that it does not provide a means of positive location of the embryo against flow of the medium in the reverse direction, which tends to move the embryo away from the constriction; it does not provide ready means of gas exchange between the medium and an external gas environment, and does not provide a ready means of storage of a number of embryos in individual locations while tracking their identity—i.e. it is possible in the apparatus and method of U.S. Pat. No. 6,193,647 for the embryos to move from one retention position to another, so losing information as to their identity. No adaptation is disclosed which will make the apparatus suitable for use in transportation, in which potential problems of the embryos moving under gravity or motion will arise.
Campbell, et al. U.S. 2002 0 068 358 have proposed an apparatus for embryo culture which is adapted for transportation, in which the embryo is retained in a well which is capable of being closed in such a way that the embryo is positively retained, and which has a supply of medium and flow generating means which allows the medium in the well to be replaced under remote or automatic control. U.S. 2002 0 068 358 also discloses means to monitor and/or control parameters in the medium or the well, such as temperature, pH, and chemical constituents, though details of the apparatus showing exactly how this is to be achieved are not disclosed. The apparatus and method of U.S. 2002 0 068 358 are poorly adapted to shipping a number of embryos in a controlled chemical environment while keeping track of their identity—there is no means of segmenting embryos in a common well or wells; the well is considerably larger than the embryo, so giving poor control of the medium environment and a long time and large volume of medium for complete exchange of a first medium for a second; access to the well is down a long inlet tube or by entrainment in a microchannel and cannot readily be achieved using conventional pipettes; the design is not suitable for use with conventional microscopy.
Thompson et al., U.S. Pat. No. 6,673,008, disclose a method and apparatus for culturing of embryos in which the embryo is cultured in medium in a tank, the tank being supplied with medium from one or more reservoirs, and optionally provided with sensors for, for example, temperature, pH, dissolved O2, ions in solution or metabolic products from the respiration of the embryo, allowing the medium around the embryo to be changed in response to conditions in the medium or to a programme stored in a control unit. The apparatus as disclosed in U.S. Pat. No. 6,673,008 comprises macro-scale devices enclosing a significant volume of solution, and the tanks of the invention are of large volume (10-50 ml), so requiring an even larger volume of medium in order to replace a first medium with a second. The device is not self-contained, in that it uses separate reservoirs and flow system components external to the apparatus and is not adapted for transportation. No means of gas (CO2, air) perfusion of the embryos inside the tank is disclosed, except by means of flow of newly gas-enriched medium from the reservoir. In a practical transportation apparatus, the size of the apparatus and hence the volume of medium surrounding the embryo is advantageously smaller than specified in U.S. Pat. No. 6,673,008, and so a means to allow gas equilibration with the medium around the embryos is preferred.
Van den Steen et al., U.S. 2004 0 234 940, disclose a micro-chamber arrangement for development of embryos that allows flow of medium through a chamber based on a stacked array of sieve-like components that retain embryos in individual compartments. The embryos are located in the compartments and the stack of sieve-like components is then assembled to enclose them. The compartments are illustrated as being approximately embryo-sized, but the illustration in US 2004 0 234 940 is purely schematic and no means is disclosed of fabricating such a structure. No lid or other means of closure is disclosed that will allow transportation of the apparatus.
Vajta et al. WO 0 102 539 disclose a method of culturing embryos in an array of small wells located at the base of a larger well (known as the well-of-wells method). This allows embryos to be located separately in a common medium, but does not include means to retain the embryos in situ if the medium or the device comprising the well is disturbed. Consequently it is unsuitable for transport of embryos outside the laboratory environment. Also, as the method is based on an open well, it relies on exchange of gas from, and heating by, the environment in an incubator. Further, no means is disclosed of changing the composition of the medium other than by pipetting the medium into and out of the larger well.
Vajta et al. U.S. Pat. No. 6,399,375 disclose transport of ova or embryos in capillary-like straws, as used for embryo transfer, the straw having optionally sealed ends, and in which the maturation or culture process can take place during transport, but this does not allow for exchange of medium during transport.
Transport devices for embryos or ova are known, for example as manufactured by Cryologic Pty (Australia) (www.cryologic.com, www.biogenics.com) which maintain constant temperature during transport over a period of hours or days, but which can not maintain a constant gaseous environment for exchange with medium in the inner containment. The inner containment is typically in the form of vials, straws or capillaries and again there is no means for exchange of medium during transport.
A further problem with devices of the prior art disclosed in U.S. 2002 0 068 358, U.S. Pat. No. 6,673,008 and U.S. 2004 0 234 940 is that they are not adapted to be small or of low aspect ratio (such as for example straws), so requiring increased volume to contain them with consequently increased power and insulation requirements to maintain their conditions during transport. This leads to the shipping time being limited and so the contents are vulnerable to delays in shipping. Additionally, apparatus presently commercially available are insufficiently well insulated and are capable of maintaining temperature by heating, but not by cooling the sample, and so the embryos and ova are vulnerable if they encounter prolonged periods of high ambient temperature.
In the following the terms ‘cellular entity’, ‘object’ and ‘embryo’ are used interchangeably for an ovum, embryo or other cellular entity that is located within the apparatus and used in the method of the invention. Relevant parts of the apparatus can be sized according to the typical dimensions of the object to be housed. Cells other than embryos, ova and the like will be smaller, and the embodiments of the invention apply to these also given the relevant parts are sized accordingly.
According to a first aspect of the invention, there is provided a device as specified in claims 1 to 23.
According to a second aspect of the invention there is an apparatus as specified in claims 24 to 35.
According to a third aspect of the invention there is provided a transport module as specified in claim 36 to 38.
The device, apparatus and module of the present invention, among other things, allows transportation of cellular entities in a reproducible and stable environment without the need for regular operator intervention.
Mention herein with regard to the flow of fluid between wells can also relate to the diffusion of chemical species/molecules therebetween.
In one embodiment, the invention provides an apparatus for culturing or maturing of cellular entities, the apparatus comprising: a device comprising a base with one or more wells opening to a surface of the base, a lid which acts to close each well against entry or exit of a cellular entity, permeation means to allow transport of molecules to the medium in the well(s) from a gas supply within the apparatus.
According to a further embodiment, the invention provides an apparatus for culturing or maturing of cellular entities, the apparatus comprising: a device comprising a base with multiple wells opening to a surface of the base, a lid which acts to close each well against entry or exit of a cellular entity, means for chemical communication between the wells, adapted so that the cellular entities are retained in their original wells, and physical contact between cellular entities contained in adjoining wells is prevented.
According to a further embodiment, the invention provides an apparatus for culturing or maturing of cellular entities, the apparatus comprising: —a device comprising a base with one or more wells opening to a surface of the base, a lid which acts to close each well against entry or exit of a cellular entity and means to modify the composition of the medium in a well while the lid is in place.
According to a further embodiment, the invention provides a system for culturing and transporting embryos comprising the device of the invention and an appliance or transport module which operates in conjunction with the device, the appliance or module comprising: one or more fluidic reservoirs for supplying fluid to the device, fluidic connection means to effect fluidic communication between the appliance and the device, flow generation or control means to effect or regulate flow on the device, a power supply to allow operation of the device and the appliance independently of an external power supply, a control means to control operation of the device and the appliance, optionally using the output from sensors associated with the device.
The surface is preferably flat or planar. The wells preferably form a two dimensional array for ease of automatic insertion of cellular entities or microscopic examination.
Preferably the apparatus is arranged to give visibility of the embryo in a well for observation through the base, using an inverted microscope, from above, using a standard microscope, or both.
Preferably means to control the temperature of the medium in the well are provided.
Preferably one or more temperature sensors to measure the temperature of the apparatus itself or the medium in the well is provided.
Preferably one or more heat transfer means to heat or cool the apparatus itself or the medium in the well is provided.
In one embodiment all or part of the device is made from a gas-permeable but liquid-impermeable material such as PDMS. PDMS has a high solubility for gas and a low solubility for aqueous liquids and so can sustain sufficient transport of oxygen and CO2 across a suitable thickness of the material for metabolism of cellular contents of the wells. The components are sized to allow sufficient transport rate through the bulk material that respiration of the cellular contents of the wells is sustained.
In an alternative preferred embodiment, the lid or base is made from a porous hydrophobic material that supports gas transport but does not allow access of aqueous liquid into the pores. Such materials exist in several forms, but one found particularly suitable is porous sintered polypropylene, trademarked as ‘VYON’ and supplied by Porvair Ltd., Wrexham, UK. This material is structurally robust and has high gas transport coefficients.
In preferred embodiments the base is thin, to allow good optical properties when placed on an inverted microscope, and also to give good thermal contact between the contents of the wells and the lower surface of the base, so allowing close temperature control of the contents when the device is placed on a heating or cooling surface.
Preferably at least part of the surface of the lid and/or the base is hydrophilic.
Preferably at least part of the surface of the lid and/or the base is hydrophobic.
Preferably a controlled release device which acts to release substances into the well is provided. The controlled release device may be autonomous, for example time-release, or controllable, for example using an external control signal or stimulus.
Preferably one or more fluidic channels in fluid communication with the well, through which medium or gas may flow are provided.
Preferably a supply of material to be added to the medium in the well, so as to change the chemical composition of medium in the well is provided.
Preferably means to allow gaseous communication between the wells and a supply of gas, either in the environment immediately surrounding the apparatus or supplied via a further fluidic channel is provided.
Preferably a gas reservoir in fluidic communication with a permeation means located on the device or as part of the fluidic flow system of the appliance, which permeation means allows transport of gas molecules from the gas reservoir to the medium in the device is provided.
Preferably thermal insulation is provided between the device and the environment.
Further temperature sensors preferably are provided that measure the temperature of the appliance or its external environment, the output of which is logged or utilised by the control means, for example to control heat transfer means or flow within the appliance or device.
Preferably at least one further sensor, for example dissolved oxygen and/or pH sensors, which monitors conditions either in medium in the well or in medium in fluid communication with it is provided.
Preferably one or more of the following is provided: —data logging means that records data from the sensors of the system, such as the temperature, pH, dissolved oxygen or other sensors as described above associated with conditions in the medium to which the cellular entities are exposed; sensors elsewhere in the system, such as internal and external temperature sensors which measure the correct functioning of the system and the environmental conditions in which it is located; accelerometers and attitude sensors which might be provided to detect motion or untoward events; communication means that allows communication between the appliance and a remote system, such as a mobile telephony interface or a wireless data interface; GPS position monitoring means; which together can act to monitor or control the operation of the appliance and the device, log its position and report status and positional information to a remote station.
The aforementioned preferred features may be provided as part of the device or as part of the apparatus or appliance.
In a further embodiment, the transport system of the invention further comprises means for stabilization of the temperature of the inside of the apparatus and or the device, comprising:
a thermally insulating outer housing comprising a receiving region for a heat sink such as a cold body
In a preferred embodiment the heat sink comprises a cold body, comprising a material or assembly which may be cooled before introduction into the apparatus.
In another embodiment the heat sink comprises a heat exchanger which acts to dissipate heat to the outside of the outer insulating housing.
In a preferred embodiment the device and heat supply means are located within a closed thermally inner insulating region outside which the cold body is located.
In a preferred embodiment the cold body is distributed substantially around the inner insulating region.
In a preferred embodiment the cold body comprises a phase change or eutectic material, for example a gel, which is adapted to absorb or release latent heat at a temperature below that at which the device is desired to be held.
The device heater and control means regulate the amount of heat needed to keep the device at a set temperature above the temperature of the cold body. The power input to the heater is controlled by the control means in relation to the rate of heat loss through the insulation to the cold body.
In an alternative embodiment, the cold body may be any other material which is suitable to be pre-cooled in a freezer or refrigerator, and which can be mounted into the apparatus before shipping. Such a material may be liquid or solid, preferably contained within a subcomponent designed for ready handling and ease of mounting in the apparatus.
In a preferred embodiment the system additionally comprises means to monitor and the temperature of the region in which the device is to be placed, before and after the cold body has been mounted in the device, to ensure that the device experiences a controlled temperature profile.
In a preferred embodiment the apparatus comprises one or more temperature sensors which sense the temperature of the cold body and which are read by the control means. The output from this sensor may then be used to monitor the status of the transport module and to control the heating supply means.
In a preferred embodiment the control means comprises a program which acts to:
sense the temperature of one or more of: the transport module, the environment outside the outer insulating housing, the region inside the outer insulating housing, the region inside the inner insulating housing, the device, the medium within the device, any reservoirs for medium that are provided within the apparatus, and the temperature of medium within the apparatus.
The wells for the cellular entity can be of any form provided that they form a designated area for retaining the cellular entity.
The invention will now be described, by way of example only, with reference to the accompanying schematic figures, in which:—
Optionally the base and lid are held together without retaining devices, for example by means of tight interfitting or adhesion between regions of the lid and the base.
The device 10 itself may be of any size, suitable to accommodate any number of wells 20. The device is advantageously formed to a standard size to interact with standard biotechnological equipment, such as microplate handlers or microscope slide holders.
The wells 20 may be sized to contain a large volume of medium per object as in
In preferred embodiments the wells 20 are sized to accommodate the objects of interest, while containing an amount of medium that is small compared with similar apparatus of the prior art. Typically the wells will have a volume between 1E-6 μl and 100 μl, and a typical minimum dimension in the range 10 μm to 5 mm. More preferably, for objects such as embryos, oocytes and cumulus-oocyte complexes, with typical dimensions in the range 50 to 500 μm, the wells will have a volume in the range 1E-3 μl and 100 μl and a minimum dimension in the range 100 μm to 5 mm. For culture of large numbers of other cells in common medium space these dimensions will also be suitable, but for culture of smaller numbers of cells the preferred dimensions are smaller, with well volume in the range 1E-6 μl and 1 μl, with minimum dimension in the range 10 μm to 1 mm.
In preferred embodiments the base 12 is thin, to allow good optical properties when placed on an inverted microscope, and also to give good thermal contact between the contents of the wells and the lower surface of the base, so allowing close temperature control of the contents when the device is placed on a heating or cooling surface.
In use, the well 20 is filled with medium 30 and objects 24 deposited into it, either manually or using a robotic pipettor. Multiple wells 20 may be formed to a standard format and arranged on a standard grid, such as the SBS microwell plate standard, to allow easy interface to robotic pipetting equipment. The base and lid are adapted to allow easy application of the lid to the base without trapping an air bubble in the well. This can be done as in standard practice using microscope slides and cover slips by arranging for at least part of the surface of the lid and base to be hydrophilic, so allowing the medium to wet the surface and a sliding motion to displace excess medium over the surface of the lid and base before they seal. In a preferred embodiment shown in
The clip means 16 is shown as a simple spring clip in
The device of
In a further embodiment, the material in regions 42 is chosen to be active, i.e. to change over time and/or in response to its environment. For example, the material is chosen from the group of slowly-hydrating hyrodgel polymers, whose diffusional properties change with hydration, the diffusion coefficient increasing with increasing degrees of hydration. In this embodiment the wells are initially isolated one from another, and are increasingly diffusionally connected as time goes on. This is potentially advantageous in circumstances where the conditions are intended to change during transportation, from culture of isolated objects to joint culture, and in particular when the composition of the medium is being changed to progress culture while in transit. Similarly, a slowly-dissolving material in the regions, such as a less cross-linked gel composition, would open the diffusional pathway over time. Provision of a hydrogel layer that at first only partially fills the region 42, but which swells gradually with time, would steadily restrict diffusional interconnection should that be desired.
In an alternative embodiment the temperature sensors are provided mounted on or associated with the base 12. The sensor might be located on the surface 18 of the base, or within the material of the base at a short distance from the bottom of the wells or the space 40.
Further, one or more temperature sensors 68 could be mounted on the base or lid of the device, so monitoring its outside temperature. If the device is located in use in a closed, insulated environment then this can be designed to be effectively isothermal, and the external device temperature will be a good approximation to the temperature of the medium.
Further, or in the alternative, one or more fluid flow passages are provided either wholly or partially defined by the material of the base and/or lid, through which fluid may flow to maintain the temperature of the device. For example, fluid flow passages may be defined within the base material as indicated in cross-section at 70 in
The above embodiments serve to retain single or groups of objects in fixed locations in controlled volumes of medium with optional diffusion between the objects. In further particularly preferred embodiments the device is adapted to change the composition of the medium bathing the objects as a function of time or in response to an external stimulus.
Other designs of release structure will be apparent to those skilled in the art and may be used in the device and method of the inventions. In particular, the substance to be released can be covered wholly or partly by a barrier material, such as for example a hydrogel, which slowly expands on contact with a liquid to become permeable.
The device 200 comprises a base 202 and a lid 208, the base optionally being formed from a substrate 204, a first body part 205 and a second body part 206 permanently bonded together. The base comprises a well 20 as before adapted to contain an object 24. The lid 208 is removable to give access to the well and when in place seals a fluidic path through the device, comprising an inlet port 210, an inlet channel 212, the well 20, an outlet channel 214 and an outlet port 216. The inlet and outlet port are shown in
The device 200 of
This form of construction has the advantage that the resulting device is optically transparent and provides observation through a good quality planar substrate using an inverted microscope.
In preferred embodiments there are multiple wells and associated flow systems as part of the device, in which case
The appliance 50 comprises fluid supply and flow means for operation in conjunction with the device, comprising one or more fluid reservoirs 306, pump means 308, waste reservoir 310, the reservoirs being equipped with breathers 312, 314 to equalise pressure. More than one reservoir 306 may be provided, each with a different medium, either connected in series in the flow path so that the contents of one flows substantially completely through the flow path before the contents of the other starts to flow through the path, or with valve means to select which reservoir is connected to the flow path. The pump then flows medium through the flow path and exchanges medium with wells 20 according to a pre-set programme or to conditions detected in the device or in the appliance. The appliance is preferably thermally stabilised using an internal temperature sensor and heater; in particular, the fluid reservoir 306 is preferably insulated and thermally stabilised to create controlled temperature conditions in the fluid flow, and so is provided with for example a temperature sensor 320 and a heater block 322. A control means 340 detects outputs from the sensors and controls the heaters to maintain a pre-set temperature or temperature profile in the wells, and controls flow of medium according to a pre-set programme.
The appliance comprises an insulating enclosure 402 that contains the device and either the whole or other parts of the flow system. The insulating enclosure is openable to insert the device 400 and may comprise more than one insulated compartment whose temperatures are either jointly or separately controlled by heat exchange means 322. The device 400 is mounted on a heat exchange block 52 as before, equipped with a heater 318 and a temperature sensor 316, though the heat exchange block might be capable of cooling also, so comprising a peltier device coupled to a heat sink, or a block comprising channels for circulating cooled fluid to and from a refrigeration unit integrated as part of the system (not shown). The flow system comprises one or more reservoirs for medium, 404, 406, a pump 308, inlet flow line 408 and outlet flow line 410 with fluidic connections to the ports of the device, and a waste reservoir 412. The pump may be on the inlet side of the device or on the outlet side as shown dotted at 414.
A preferred embodiment of the flow system for the system of the invention is shown in
The insulating housing 402 may also be gas-tight, so as to contain a gaseous atmosphere for gas exchange with the medium in the reservoirs, the device or both. The reservoirs may therefore be provided with breathers to assist this process, the breathers being made for example from a porous hydrophobic polymer. The breathers may alternatively vent to the external atmosphere. The valves 422 and 426 are in preferred embodiments replaced by manual sealing caps in the ports 420, 424, arranged to be sealable without trapping air in the reservoirs.
The system of
Other configurations of the device, appliance and flow system are envisaged for use in the system of the invention. For example, a flow system as known in the art, where a number of reservoirs are connected to a common flow line and flow controlled by valves associated with each, or separate pumping means associated with each, might also be used. Pumping means for the system include displacement pumps, pressurization of the medium either by gas pressure within the reservoirs or by deformation of the reservoir walls by mechanical actuation or external fluid pressure, or any other means known in the art.
The reservoirs, pump means and other flow components may be integrated onto the device itself, or the device and all or part of the flow system might be integrated into a subassembly which itself interfits with the transport module or appliance and remaining parts of the system.
In a further embodiment the device additionally comprises a memory such as a microchip-based, or magnetic strip-based, memory system that allows data about the device and its contents to be recorded, read, stored, transported along with the device. In a preferred embodiment the memory and associated control circuitry is mounted on or within the device, together with a power source where needed. The memory system may be connected to other systems off the device by means of electrical contacts, wireless or optical communication, or it may be recorded and read magnetically. In a preferred embodiment the memory system contains information about the identity, history, contents, next actions and operational information concerning the objects and media in use on the device.
The memory system might comprise a device control system which acts to control functions on the device either independently of, or together with, the control system of the appliance, for example to indicate the status of objects in particular wells on the device and to prompt or prevent intervention by a user in the case of the whole device, objects in all, or in just some of the wells.
In a preferred embodiment the device is operable in conjunction with a further control means associated with observation of the objects on the device, for example by microscopy, in which the microscope control means is able to read from or write to the memory on the device, details of the objects in the wells of the device, media conditions, experimental observations and instructions for next actions either by the system comprising the device and the appliance, by a future experimenter, or both. In preferred embodiments the memory system of the device interacts with a laboratory information system to control the use and operation of the device and/or the appliance so as to track the use, record the conditions, or ensure compliance with record keeping or other regulatory activities.
The above embodiments require the mounting onto or within the device of an electronic system, examples of which are known in the art, and the provision of electrical contacts as disclosed for several of the embodiments above. Alternatively, wireless communication may be made between the device, the applicant or another off-device system. In either case the design required to mount the memory system on or within the device is standard and known in the art.
In a further embodiment the appliance additionally comprises one or more of the following:
data logging means that records data from the sensors of the system, such as the temperature, pH, dissolved oxygen or other sensors as described above associated with conditions in the medium to which the embryos are exposed;
sensors elsewhere in the system, such as internal and external temperature sensors which measure the correct functioning of the system and the environmental conditions in which it is located;
accelerometers and attitude sensors which might be provided to detect motion, shock or untoward events;
communication means that allows communication between the appliance and a remote system, such as a mobile telephony interface or a wireless data interface;
GPS position monitoring means;
which together with the control means of the appliance can act to monitor or control the operation of the appliance and the device, log its position and report status and positional information to a remote station.
It is useful in the case of loss or delay in transport to be able to locate the transport system of the invention and optionally to receive information on its status and the status of the objects within it. The above features allow this to be done.
A system is provided for transporting embryos comprising a device having wells for the embryos, the wells being closed by a lid, and a transport module or appliance as described above acting to:
control the temperature of the embryos,
It is an object of the invention to provide an apparatus and method for transporting a payload at a controlled temperature, in which drawbacks in the apparatus of the prior art are overcome. Such drawbacks include: poor temperature regulation; short endurance before temperature drifts out of specified range; large size and/or weight to achieve endurance of the order of 4 days or more; tendency of cool transport apparatus, intended to maintain temperatures close to 0 C, to freeze the sample when this is first loaded into the apparatus and compromises to the performance of the apparatus introduced to counteract this; and lack of ability of warm transport apparatus, intended to maintain temperatures above mean ambient, to resist over-temperature for extended periods. Prior art apparatus all suffer from at least one of the above problems. Mean ambient temperature is defined in the following as a mean temperature in the range approximately 10-25 C.
Nagle U.S. Pat. No. 6,020,575 discloses apparatus intended for shipping at above mean ambient temperature, having an outer insulation layer defining an inner space, with an electric heater and a eutectic material (or “Phase Change Material”, PCM) together closely adjacent in the inner space, the eutectic material intended to assist in the heating action.
Rix U.S. Pat. No. 6,822,198 discloses a transport apparatus comprising an insulating housing enclosing an inner electric heater and a cooling pack. The position of the cooling pack relative to the heater is not disclosed, and there is no insulation between the heater and the cooling pack. This apparatus has no feature to prevent contact between the cooling pack and so potentially suffers from uncontrolled heating of the cool pack by the heater, and so in use will have variable and potentially short endurance; also, uncontrolled temperature gradients will exist within the chamber between the heater and cooling pack.
Nadeur WO03/101861 discloses a shipping device including a body comprising PCM surrounding and in contact with a payload, the PCM having a melting point Tc substantially the same as the storage temperature for the payload. This device will keep the temperature stable once the PCM has reached Tc, but in order to freeze the PCM it needs to be cooled some way below Tc. In order to warm the PCM to Tc, it needs to be conditioned, i.e. warmed, which takes time, is prone to error, and owing to the extended range of melting which many PCM have, wastes a considerable portion of the cooling capability of the PCM. Especially for apparatus operating close to 0 C, there is a danger of an aqueous payload freezing, which is to be avoided for biological samples.
No transport apparatus is known in the prior art that combines high capacity coolant with the ability to use a conventional freezer at −15 C to −20 C to freeze the coolant, in a design which will substantially prevent a payload cooling below 0 C, while providing a interior temperature close to 0 C.
Temperature controlled transport apparatus operating at temperatures above mean ambient are known, for example to transport living biological samples at the temperature range 37-39 C. These apparatus usually rely on insulation and an inner heating means, for example pre-heated PCM or an electric heater powered by a battery pack, and have an endurance that is limited by the capacity of the battery or PCM and by the insulation. The apparatus of the prior art adapted for small scale transport of biological materials have no refrigeration capability however, and so are liable to overheating in high ambient temperatures, such as are likely to be encountered in the course of shipping in warm climates.
Over-temperature protection for temperature-sensitive goods is disclosed by H of et al. U.S. Pat. No. 4,425,998, which provides a layer of PCM in the form of a salt with a melting point Tc just below the sensitive temperature of the goods, surrounded by an insulating outer housing. The arrangement of H of et al. is not suitable for protection of a heated payload, however, as heat flux from the payload (which is above Tc) will tend to melt the protection salt.
The further the melting point of the salt is below the operating temperature, and the better is the external insulation, so the better is the thermal protection, but the greater is the tendency of the salt to be melted by the heated payload. The present invention differs from the design of H of et al. by providing an inner insulation layer and by selecting advantageous combinations of the insulation and PCM parameters.
The invention provides an apparatus for transporting a payload at a controlled temperature, comprising an outer housing, outer insulation region, a heat sink region comprising a heat sink such as a heat absorbing material, in some embodiments a heat sink component such as a cold body, which may be pre-cooled before introduction into the apparatus; an inner insulation region and a heated payload. The apparatus can be adapted to operate at any required temperature in the range from below zero to significantly above mean ambient temperature.
In a first preferred embodiment the apparatus is adapted for use at above mean ambient temperature, such as in the range 37-41 C, for example for incubation and transport of cellular entities such as cells in culture, embryos or oocytes; in this embodiment the heat sink is preferably in the form of a heat absorbing material and acts to protect the apparatus against over-temperatures resulting from prolonged exposure to high ambient temperature.
In a second preferred embodiment the apparatus is adapted for use at below mean ambient temperature, such as in the range 0-10 C, for example for transport of tissue samples, organs, blood or blood products or temperature-sensitive pharmaceuticals or other chemicals; in this configuration the heat sink is advantageously in the form of a heat absorbing material in one or more containers that are reversibly removable from the apparatus, and may be cooled before being introduced to the apparatus.
In either of the above embodiments the heat absorbing material is preferably a phase change (PCM) or eutectic material that has a transition temperature lower than the desired control temperature of the payload. In the higher temperature case, the PCM preferably has a transition temperature in the range 10 C-1 C, more preferably in the range 5 C-2 C; i.e. allowing a small margin of temperature protection below the desired operating temperature. In the lower temperature embodiment slight over-temperature is most likely less important, so the PCM preferably has a transition temperature in the range 10 C-0 C below, more preferably in the range 4 C-1 C below the desired operating temperature. A particularly preferred embodiment for use in the range 1 C to 4 C uses a water-based heat absorbent material with a transition temperature close to 0 C.
The payload unit comprises an inner housing 518, which holds a payload 520, a heater unit 522 which heats the payload, a control means 524 and a power supply (for example batteries) 526. The control means measures the temperature of the payload by means of a temperature sensor 528. In some embodiments the sensor 528 is mounted on the payload itself; in other embodiments the payload is housed in a payload container (not shown in
In preferred embodiments one or both of the insulating regions comprise one or more vacuum insulation panels (VIPs). The outer housing may additionally comprise insulating and/or shock absorbing material, for example expanded polystyrene (EPS).
In use the heater controls the payload temperature against heat flux to ambient. When the ambient is below the control temperature heat is lost through the inner insulation, the heat absorbing material and the outer insulation. The heat absorbing material is chosen to have a higher heat capacity than the insulation and acts to buffer the heat flux to/from ambient by absorbing and giving out heat. In the case that the heat absorbing material is a PCM, the PCM acts as a thermal reservoir at or near the transition temperature. The operation of the apparatus is illustrated by the example that the control temperature of the payload is 38 C, suitable for culture of embryos. The particular advantage of the apparatus in
The endurance at ambient temperatures above Tc depends on the heat capacity of the heat absorbing region and the thermal resistance of the outer insulating region, and these are chosen to give an advantageous compromise between protection and size and weight of the apparatus. The sum of the thermal resistances of the inner and outer insulating regions determines the power requirement of the heater and the endurance of the apparatus for given battery capacity at low ambient temperatures. In the case that the heat absorbing material is a PCM, for over-temperature protection to work the PCM should be substantially frozen when the apparatus is in a normal temperature ambient. In preferred embodiments of an apparatus operating at 38 C, with a PCM with Tc around 35 C, the outer insulating region preferably has a lower thermal resistance than the inner region. This means that the PCM is poised closer to mean ambient temperature than 38 C, so keeping it frozen. However, the less the outer-thermal insulation, the greater the heat capacity of PCM that is needed to maintain protection against over-temperature. For preferred embodiments in which the inner and the outer insulation comprises VIPs, the tradeoff is between thickness of VIP and thickness (and mass) of PCM. In a typical embodiment of the apparatus, adapted to operate at a control temperature in the range 37-40 C, preferred ratios of thickness of the inner to outer insulation are between 1:1 and 4:1. For embodiments designed to operate at control temperatures closer to mean ambient temperature, the optimum ratio will be different: Tc of the PCM will be lower, and a greater proportion of the total insulation is advantageously placed outside the PCM to slow heat conduction to the PCM in over-temperature conditions. The ratio of outer to inner insulation is chosen according to the design requirements of the apparatus.
In a typical embodiment of the apparatus, adapted to operate at a control temperature in the range 37-40 C, using VIPs of thermal conductivity 0.0042 W/mK (Vaq-VIP from Va-Q-Tec GmbH, Wurzburg, Germany) and PCM with Tc 35 C and latent heat capacity 99 kJ/litre=500 kJ/m2 for 5 mm thick panels (Rubitherm RT35 in fibreboard form, Rubitherm GmbH, Hamburg, Germany), the ratio of thickness of the inner to outer insulation may be chosen to be between around 1:1 and around 4:1. Examples of preferred embodiments are given, but no limitation to these is to be understood. Preferred embodiments using these materials have outer VIP in the range 5-15 mm thick, PCM layers in the range 5-10 mm thick and inner VIP layers in the range 1 to 4 times the thickness of the outer VIP. A preferred embodiment has an outer insulation VIP approximately 5 mm thick, a PCM layer 8 mm thick and an inner VIP approximately 20 mm thick. This combination will give over-temperature protection for a transport appliance with a control temperature of 38 C against 50 C ambient for around 8 hr. A further preferred embodiment has an outer insulation VIP approximately 8 mm thick, a PCM layer 5 mm thick and an inner VIP approximately 17 mm thick. This combination will give also over-temperature protection for an apparatus with a control temperature of 38 C against 50 C ambient for around 8 hr.
In the embodiment in
The apparatus may be of any desired shape (though is most easily fabricated with rectangular faces) and may be fabricated from a variety of materials and in a variety of ways as known in the art. The insulation regions are preferably formed from VIPs, either discrete panels for each face of the apparatus or one or more continuous panels formed to fit the outer housing. Examples of suppliers of suitable panels are ‘Va-Q-VIP’ from Va-Q-Tec GmbH, Wurzburg, Germany; ‘VacuPanel’ from Technautics Inc., Costa Mesa Calif., USA; VIP (unbranded) from ThermoSafe Inc., USA. The VIPs are preferably protected by thin protective layers or liners (not shown in
In a preferred embodiment the inner insulated housing 542 is gas-tight, so allowing a different gas atmosphere to be maintained in the space 514 from in the rest of the apparatus. This is advantageous for example if the payload contains cells in culture, embryos, oocytes etc., in media which require a CO2 atmosphere for pH control. In this case, the lid 544 of the inner insulated housing preferably has a gasket or O-ring pressure-tight seal to the base of the inner housing. Gas inlet 558 closed by valve 560, and gas outlet 562 closed by valve 564 are provided to introduce a gas atmosphere into the inner housing 542. Power line connector 556 is adapted to be gas-tight also.
In the embodiment in
Gas inlet 558, inlet valve 560, outlet 562 and outlet valve 564 are provided to allow gas to be flowed into the space from outside the apparatus once the apparatus is closed. In a preferred embodiment, housing 518 is closed by a gas-tight lid 586. In some embodiments lid 586 defines an upper payload space 522 which may be separate from the main payload space 514. In
The embodiment in
In this and previous embodiments the heating means is preferably an electric heater. In an alternative embodiment the heating means comprises a fluidic heat conducting means which acts to heat the payload from a heat source, for example an electric heater, elsewhere in the apparatus, for example by means of fluid flowing through heating channels in the body of a microfluidic device 570.
The payload container shown in
In a preferred embodiment, adapted for use in the temperature range 0-10 C, for application for example in transport of tissue samples, the heat sink components 610 comprise a water-based coolant. The components may take the form of bottles, adapted to fit into the heat sink region in space 609, or in alternative embodiment may be conventional gel packs in flexible packaging and frozen in a shape that allows them to fit into the heat sink region.
In use in preferred embodiments the heat sink components are frozen in a conventional freezer and may be placed in the insulated housing straight away from the freezer. The inner unit, comprising the payload, heater, control means and power supply, may have the batteries charged while outside the apparatus, and pre-set using controls on the inner unit to the desired temperature, then inserted into the apparatus adjacent to the heat sink components. The sensor 628 detects the fall in temperature resulting from conduction through the inner insulating region 612 to the heat sinks, and the control means heats the payload space to maintain the desired temperature against cooling from the heat sinks. The lid 604 is fitted, and the apparatus may now be shipped. Once the heat sinks reach around 0 C the temperature remains nearly constant—the heater then runs to maintain the differential between the control temperature and 0 C. For low control temperatures, e.g. 2 C as appropriate for tissue samples, only very low power is needed to do this, as a result of the inner insulating region. Prior art transport systems which do not have such an inner insulating region have a much higher power requirement, with consequent short endurance from a given battery capacity, and rapid loss of cooling capacity. Outer insulation 608 serves primarily to insulate the coolant from melting; the inner insulation controls the temperature gradient between the payload and the heat sinks 610.
A great advantage of this embodiment is that a sample can be kept close to 0 C without the danger of freezing and consequent degradation of the sample. Also, compared with transport apparatus of the prior art in which payload temperatures are kept above 0 C by buffering with water at 4 C or using PCM with transition temperature above 0 C, the apparatus of the invention has a much longer endurance for a given size and weight. The water used for buffering contributes little cooling capacity per unit volume and mass; the PCMs with transition temperatures at say 4-6 C have both lower specific latent heat and lower density, so having a latent heat per unit volume as low as half that of water. Additionally, pre-conditioning (partial thawing) of the coolant, necessary even when using PCM with Tc above 0 C in prior art non-heated transport apparatus, is not necessary, so avoiding a significant source of potential failure in the transport protocol.
Control temperatures significantly above 0 C may be achieved with the embodiment above, at the cost of increased power required for the heater. Preferred embodiments for operation at significantly above 0 C may have more insulating inner insulation regions 612. In preferred embodiments a PCM is used in the heat sink components that has a Tc value within a limited temperature range at or below the desired control temperature, in order to minimize the battery capacity needed for a given shipping endurance. For example, in a preferred embodiment adapted to run in the temperature range 8-15 C, a phase change material with Tc at 4 C-8 C may be used instead of ice, and for the range 10 C and above, a phase change material with Tc in the range 5 C-10 C may be used. In general in preferred embodiments a PCM is used that has a Tc around 0 C-20 C below the control temperature, in more preferred embodiments 1 C-10 C and inmost preferred embodiments 1 C-5 C.
In preferred embodiments the outer insulation comprises at least one VIP panel, and in more preferred embodiments one VIP panel for each face of the apparatus. The insulating properties of the VIP are chosen with regard to the intended endurance of the shipper in given ambient conditions. In some embodiments VIP panels are use also for the inner insulation region. In preferred embodiments the requirements for the inner insulation are less strenuous than those for the outer insulation and so other insulation materials, for example structural polymer foam, may be used. The inner unit may be housed in a structural housing (not shown) if required.
An experimental apparatus of the embodiment of
A thin 50 W sheet-form heater was mounted on the inside of the inner insulation, around the payload container 620, and a heater control means set to a control temperature of 1 C was connected with a temperature sensor adjacent the heater as shown as 628. An ‘i-button’ temperature logger was placed on the inside of the payload container. The mean ambient temperature was around 20 C. The frozen ice packs were placed in the heat sink region at −18 C. The temperature inside the payload container had reached 1 C in around 10 hr and remained within 0.25 C of 1 C for an endurance of greater than 7 days (at which time the test was terminated). Total energy consumption over 7 days was 2.5 kJ (mean power 4 mW). For comparison, in experiments using the same housing, outer and inner insulation but without active heating, using PCM with Tc 4-6 C and specific heat capacity 2.4 kJ/kg and relative density 0.8 filling the containers, inserted into the apparatus at −18 C, the payload temperature fell below 0 C within 3 hours. Used without electric heating ice-based gel packs, with specific heat capacity of 4.2 kJ/kg, would be expected to cool the payload below 0 C in an even shorter time. Using the Tc=4-6 C PCM the payload temperature rapidly reached 4 C and drifted steadily upwards to reach 8 C after 4.5 days, beyond which the PCM had melted completely and the temperature rose rapidly. The apparatus of the invention had better short term resistance to freezing, better temperature regulation, and much longer endurance for a given size and weight than the comparable apparatus without the configuration of the invention.
Alternative embodiments to that in
In an alternative embodiment (not shown) the heater or conductive component(s) may be shaped to interfit with the payload container or the payload itself to give good thermal contact between them. For example, the heater or conductor may be in the form of a rod onto which the container fits, so giving a radial heat flux from the centre of the payload container, outwards to the inner insulation and thence to the heat sinks.
Temperature sensor 630 is optionally provided to read ambient temperature. Control means 624 and power supply 626 are separated from the payload space by inner insulation 610 and from the heat sink components 610 by the partition 654 which in preferred embodiments is itself insulating to prevent heat from the control means and power supply reaching the heat ink components. The location of the power supply outside the inner insulation in this embodiment is advantageous where the power supply dissipates more heat, especially while charging the batteries, than can conveniently be lost through the inner insulation. In some embodiments parts of the power supply (such as power transistors or ICs) might be arranged to be in good thermal contact with the outer housing to allow dissipation while charging batteries.
In the embodiments in
It will be understood that by using a PCM with a different Tc, an apparatus adapted for a different range of control temperatures can be constructed. For example, PCM at the following temperatures is known to be available: −4, −1, 0, 2-6, 3-9, 5, 7, 20-22, 24, 26-28, 29, 32, 33-38, 35-36, 44-45, 48, 58. Apparatus suitable for use at control temperatures in the range 0-20 C, preferably 1 C-5 C above Tc can be fabricated and achieve temperature control using electrical heating to raise the payload temperature above Tc. In each case, the presence of an inner insulation layer between the PCM and the heater is essential to give optimum performance.
It will be understood that in the embodiments above the control means may be of any kind known in the art. In preferred embodiments, the control can communicate with external devices to upload programs, download data, give status updates etc., by means known in the art including RF, IR, Bluetooth, USB or other cabled connection.
In a further embodiment the apparatus additionally comprises one or more of the following:
data logging means that records data from the sensors of the system, such as the temperature, pH, dissolved oxygen or other sensors as described above associated with conditions in the payload;
sensors elsewhere in the system, such as internal and external temperature sensors which measure the correct functioning of the system and the environmental conditions in which it is located;
accelerometers and attitude sensors which might be provided to detect motion, shock or untoward events;
communication means that allows communication between the appliance and a remote system, such as a mobile telephony interface or a wireless data interface;
GPS position monitoring means;
which together with the control means of the apparatus can act to monitor or control the operation of the apparatus and the device, log its position and report status and positional information to a remote station.
It is useful in the case of loss or delay in transport to be able to locate the apparatus of the invention and optionally to receive information on its status and the status of the objects within it. The above features allow this to be done.